Slide 1

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 2

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 3

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 4

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 5

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 6

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 7

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 8

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 9

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 10

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 11

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 12

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 13

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 14

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 15

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 16

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 17

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 18

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 19

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 20

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 21

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 22

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 23

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 24

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 25

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 26

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 27

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 28

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 29

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 30

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 31

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 32

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 33

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 34

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 35

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 36

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 37

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 38

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 39

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 40

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 41

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 42

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 43

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 44

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 45

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 46

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 47

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 48

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 49

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 50

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 51

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 52

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 53

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 54

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 55

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 56

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 57

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov


Slide 58

Institute for microbiology presents

TRACING THE CRIMINAL

Part eight:
Acidoresistant (acid-fast) criminals

Intro: Spitoons in fight with TB
In Czechoslovakia between
World War I and World
War II the a society was
formed with personal
engagement of president
Masaryk and his „League
against TB“. It showed a
big effort in fight against
this disease. The part of
this was education for
people not to spit to the
floor, but to use spittoons.
www.bikupan.se

Survey of individual parts
Clinical characteristics of acid-fast bacteria
Special properties of acid fast-bacteria
Diagnostics of acid-fast bacteria

Clinical
characteristics of
acid-fast bacteria

• Johny did know allready for
many years that he is HIV
positive. He knew pretty well
that he is more vulnerable than
other people and that each
infection can get him more
quickly than other people.
• Nevertheless he was surprised
that.he started to cough
recently. His doctors tried
various variants, but after
roentgen, PCR examination and
culture examination came to
conclusion that it is a miliar
(granular) form of tuberculosis.

http://cs.wikipedia.org/wiki/Tuberkul%C3%B3za

Story One

http://www.genomeindia.org

The criminal was…
• Mycobacterium tuberculosis, although TB may be
caused by Mycobacterium bovis, too.
• Interesting for this microbe: it lives inside cells. This
is also related with the fact that antibody response
is weak in tuberculosis (so neither antigen nor
antibodies are detected) and cell imunity is very
important (in vaccination, too).
• As in HIV infection just cell immunity is dammaged,
TB is one of oportune infections.

Mycobacterium inside a cell

http://www.nature.com/nrm/journal/v2/n8/fig_tab/nrm0801_569a_F3.html

Tuberculosis
• At the first contact with the infection is formed primary
complex. It is a focus (usually localized in lungs) and
corresponding regional lymphonode.
• During the next infection post-primary TB is formed. It is
worse. Usually a granulomatous formation is formed,
later it subdues caseification („becoming cheese-like“)
and then it is not enlarged anymore. Paradoxically,
majority of dammage in the organism is caused by the
host organism reaction (late hypersensitivity – in the
matter of fact, a specific type of an allergy)
• After years the original focus may re-activate, mostly in
old age, at immunodeficiency, or ethanol abuse. Such
person may be very dangerous for his/her environment.

Tuberculosis

http://www.stockmedicalart.com

www.tusalud.com.mx

Pulmonary form of TB is
not the only one
sitemaker.umich.edu (2×)

http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_incidence_2011.png

TB worldwide

Once more TB
http://www.lung.ca

http://www.cbc.ca

Brain tuberculoma

http://pathology.mc.duke.edu

More special things in TB
•

•

•

•

Their cell wall is highly hydrofobic, it contains,
mycolic acids. They nearly do not Gram stain,
special staining methods are needed.
It grows slowly, its generation period is long, so
special media are needed
They are very resistant to disinfectants. It is
impossible to use „A“ class disinfectants against
common bacteria, you need „T“ (against TB),
eventually „M“ (against atypical mycobacteria)
They are also resistant to antimicrobial drugs.

www.primer.ru

Cell wall of
mycobacteria

http://oregonstate.edu

Story two
• Mr H. lived in desert part of Sudan, where wars
and unrests were very common.
• Recently even friends that still did neither run
away nor die started to dislike contacts with mr. H.
Mutilation of face of mr. H. was a clear mark, that
mr. H. suffers the disease still too common in this
part of world.
• Good luck that H. met members of a nongovernement organisation, that where sure about
diagnose. Using dapson it was possible to help Mr.
H.

Causative agent is
•

•

•

•

Mycobacterium leprae, a microbe even stranger
than TB mycobacterium.
Its generation period is much longer than in TB
mycobacteria. In vitro culture was succesfull only
recently and it durated the whole year.
Basic treatment of leprosy is not expensive,
nevertheless in countries where leprosy is
endemic it is still too much.
Therefore leprosy still requires help of
foundations, non-government and charitative
groups.

Leprosy
• Long incubation period – 2, 3, 7 … yrs

• Tuberculoid leprosy

http://blanicti-zoldneri.tym.cz/clanky/lepra.html

– Depigmented spots on skin, swollen periferial
nerves
– Active cellullar immunity
– Spontaneous consolidation

• Lepromatous leprosy
http://abicko.avcr.cz/archiv/2002/10/obsah/9-gallery.html

– Insufficient cellullar immunity
– Confluent and swallen skin lesions, eyebrows fall
out, nose is in depression, fingers are lost, people
become blind

Leprosy

www2.bc.cc.ca.us (2×)

Story three
• Mr. P. was a furious aquarist. Last month he had a
problem: he had to use his left hand only to do
anything inside the fish tank, as he had an ulcus on
his right hand.
• After examination, his case was closed as so
named fish tank granuloma, common in aquarists.
A simillar disease in swimmers is called swimming
pool granuloma.
• Causative agents are…

Atypical mycobacteria
• Besides M. tuberculosis and M. leprae there exist
also plenty of other mycobacteria. Some of them,
e. g. Mycobacterium marinum, are so named
atypical mycobacteria, sometimes causing wound
infections and other problems.
• Some mycobacteria are non-pathogenous and
they are normal part of human microflora, e. g. M.
smegmatis.

http://www.microbeworld.org/component/jlibrary/?view=article&id=5

Infection of M. marinum

http://www.nejm.org/doi/full/10.1056/ENEJMicm000083

Story four
• Mrs. F. found several months ago some small
formations in her neck.
• Recently one of them opened to skin and dense,
yelow, unpleasantly spelling pus appeared in the
orifice. So, Mrs. F. visited her doctor.
• The doctor sent the pus for microbiological
examination. Examination durated very long time
– mrs. F. was already angry that microbiologists
are lazy and don‘t want release the results. Finally
the criminal was found…

…it was actinomycosis
• So the criminal was Actinomyces sp.
http://en.wikipedia.org
• Actinomycetes are filamentous bacteria, in fact
Gram-positive, but they do not Gram stain very well,
because their cell wall is hydrofobic and contains
many mycolic acids.
• Actinomycetes are commonly found in oral cavity of
healthy persons. From here they might commonly
get to soft tissues of nect, face or thorax. They are
anaerobic bacteria (or at least they grow the best at
anaerobic conditions).
• Similar to actinomycetes are nocardiae, but they are
strictly aerobic. Otherwise they are simillar in many
ways.

Actinomycosis

pathmicro.med.sc.edu (3×)

pathmicro.med.sc.edu

Nocardiosis

http://www.asm.org

Nocardia pneumonia
with septicaemia
A previously well 57year-old man … 3-day
history of severe
dyspnoea. We conclude
that N. asteroides
infection can present as
a fulminant communityacquired pneumonia
with bacteraemia in the
absence of
immunosuppression or
coexistent infection.
(From the article
related to the picture)

Brain nocardiose
on CT

http://www.appliedradiology.com

Special properties of
acid-fast bacteria

Acidoresistance + alkaliresistance
• Acids and alkali act only to hydrofilic components,
communication to water environment. In mycobacteria
this is not fulfilled.
• So acids and alkali have weak effect only to them.
• Acids are also not able to decolorise them, when
somewhy it was possible to stain them.
• Majority of dyes is hydrofilic, too, and so mycobacteria
stain poorly, usually it is necessary to stain them at hot
temperature, to stain them at all.
• Nocardia and Actinomycetes, unlike mycobacteria, are
only partially acidoresistant. So, we stain them by Gram,
but we have to know, that they stain poorly and
inconstantely.

Consequences for clinical doctors
• Clinical doctor, sending sample (sputum, urine,
pus or anything) „for bacteriological culture“,
cannot hope in geting reference of eventual TB
infection.
• To get info about TB, it is necessary to send
sample separately and to mark it so that it should
be examined for TB (TB-culture or TB-PCR). If so,
the laboratory can perform the needed
procedures.

Diagnostics of acidfast bacteria

How to search for criminals
• Microscopy: We use Ziehl-Neelsen stain and fluorescence
stain.
• Culture: We use special media, and before the culture the
specimen should be treated by a hydroxide. The aim is to
kill other bacteria, that would be more succesfull as they
grow more quickly. Alkaliresistant mycobacteria survive
that easily.
• Automatic culture: Various types of culture automats are
used: they are able to detect culture positivity much
sooner than classic culture.
• Biochemical differenciation is possible in specialized
laboratories.
• Animal experiment: guinea pig is used sometimes.
• PCR diagnostics is more and more important.

Mycobacterium tuberculosis

Photo O. Z.

http://www.health.qld.gov.au

Ziehl-Neelsen staining
• In step 1 we stain by carbolfuchsine (Gabbet) in hot until
steam rises. Without heating mycobacteria could not be stained, except use
of more concentrated carbolfuchsin.

• In step 2 we decolorize (cca 15 s) by „acid alcohol“, what
is mixture of alcohol with a mineral acid, most commonly
HCl. After that we rinse the slide with water.
• In step 3 we counterstain the background, so everything
decolorized in Step 2. We counterstain by methylene
blue. cca 30 s (it would be also possible to use malachit
green) and we rinse the slide with water, we dry it and
we observe it with immersion objective.
• Result: red acidoresistant rods on blue or green
backround.

Ziehl-Neelsen stain

www.spjc.edu

Ziehl-Neelsen stain

http://es.wikipedia.org/wiki/Archivo:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

It is interesting,
that ZiehlNeelsen staining
may be used
also for a group
of parasites, so
called intestinal
coccidia
(cryptosporidia
and cyclospores)

Cryptosporidia
http://www.scientificdevice.com/i
ntl_product_pages/icryptosporidiu
m_stains.htm

Culture of mycobacteria
• Hydroxide should be used before culture.
• We use liquid Šula or Banić media and egg Ogawa
or Löwenstein-Jenssen media. Egg media are solid
because of egg white coagulation, they do not
contain agar.
• Even solid media are in test tube and closed firmly.
This is not only because personel would be
endangered, but also as media would dry.
• Results are read after 1 (check for contamination),
3, 6 and for sure after 9 weeks of culture. (Positive
results are mostly found after 6 weeks of culture.)

Appearance of
mycobaterial colonies

http://www.stockmedicalart.com/

To liquid Šula medium
• Even positive test tube
is clear by first view, as
the growth of
mycobacteria is visible
only at the bottom
(„blue mess“, as
student J. H. called it
)

Tests of antituberculotic susceptibility
(not antibiotic!)
• Antituberculotics are strange chemicals, different from
antibiotics (with exceptions).
• Allways we combine 3 or 4 of them: resistances appear
quickly, and some have only intra- of only extracellullar
effect.
• We cannot use diffusion disk tests.
• Antituberculotics are added directly into culture media,
growth control is added.
• Growth present  mycobacteria resistant.
• Growth absent  mycobacteria susceptible.

Survery of commonly used
antituberculotics
Antituberculotic

Abbrev.

Isoniazid
Ethambutol
Rifampicin
Pyrazinamid
Streptomycin

H, INH
E
R
Z
S, STM

Tuberculous liver of an
experimental guinea pig
Courtessy of dr. Jana Svobodová and
dr. Lev Mezenský

PCR for TB

Institute for microbiology

1, 2, 3, 4 = patients No.
1, 2, 3, 4
5 = positive control 6 =
negative control
7 = ladder (to measure
position of a band)
upper row (c) = sample
strip, lower row (b) = IC

PCR is a method used in TB diagnostics more than
diagnostics of other bacteria. The reason is that it makes
the diagnostics much faster and the risk of
environmental contamination is not so serious.

PCR kit for TB diagnostics

http://www.cinnagen.com

Indirect diagnostics of tuberculosis
• The most important type of immunity in TB is cellmediated imunity.
• Formation of antibodies occurs, but measurable
levels of antibodies are present only in some
cases. So positive finding of anti-TB antibodies is a
sign of infection, but negative finding has very low
information value.
• Cell-mediated imunity may be tested
– by skin test (tuberculin test), especially after
vaccination
– by INF-gamma release test (reaction of pacient cells to
antigen exposition is tested).

Skin test (Mantoux)
• It is used for checking of vaccination effect, but
also for proof of an eventual latent infection.
• The complete living patient is needed for the test,
so it is not a laboratory test. Test is performed by
dermatovenerology
or
other
specialized
departments. Recently they are replaced by next
type tests
• The tests are positive in case of activation of cellmediated immunity; in the matter of fact, it is a
specific type of delayed allergy.

Test of interferon gamma release
(Quantiferon© TB-GOLD)
• A modern way of checking the cell-mediated immunity is
examination of induced interferon gamma release; in
practice, the only really used test is Quantiferon TBGOLD, that is why only this test would be mentioned
later.
• It was proven that in TB, including latent TB, tuberculosis
antigens activate T-lymphocytes and they produce big
amounts of interferon gama.
• Similarly those T-lymphocytes may be activated nonspecifically by mitogene, that is why mitogene is used as
a positive control.

Quantiferon – three test tubes
• We need non-clotted (heparinized) blood to three
test tubes (we need lymphocytes!).
• First test-tube contains the mitogene (MIT) –
here, in normal circumstances, always stimulation
of IFN-gamma should be observed.
• Second test-tube contains TB antigens (TB) – here
IFN-gamma formation stimulation should be
observed in TB infection only.
• Third test-tube does not contain anything (NIL) –
here we should (normally) never see IFN-gamma
stimulation.

Quantiferon – results
• Interferon concentration is measured by ELISA
• As positive we consider a result, where Tlymphocytes
react
to
stimulation
of
mycobacterium antigen, but in test-tube with
„nothing“ the INF-gamma is not formed.
• As negative we consider a result, where Tlymphocytes react to mitogen stimulation, but
they do not react to mycobacterial antigen
stimulation.
• Unsure result is seen (1) if T-lymphocytes are not
activated by the mitogen or (2) IFN-gamma is
formed even in the test-tube where no stimulator
was present.

Results–example*
NIL
[IU/ml]

TB minus NIL
[IU/ml]

MIT minus
NIL [IU/ml]

< 0,35
≥ 0,35

≥ 0,5

and < 25% of
NIL value

≤ 8,0

> 8,0

≥ 0,5

≥ 0,35 and ≥
25% of NIL
value

Any value

< 0,35

< 0,5

≥ 0,35 and <
25% of NIL
value

< 0,5

Any value

Any value

*the result may be different
in subtypes of the test
Final test
interpretation

Negative

Presence of

M. tuberculosis
infection

Not likely

Positive

Likely

Unsure

Cannot by
determined

Nocardia and Actinomyces
microscopy
• These bacteria are Gram staining, although they
staining poorly and thay are very pleomorphous.
• Both of them are typical by their branched
filaments, staining Gram-positive, although some
parts of the filament may stain Gram-negative or
they might remain unstained at all.
• Sometimes, short (coccoid) forms may also occur
in microscopy.

Actinomyces israeli

pathmicro.med.sc.edu

Nocardia asteroides

www2.mf.uni-lj.si

http://www.nocardia.it

Nocardia and Actinomyces culture
http://filebox.vt.edu

• Although both genera are
similar in many properties,
one is different: Nocardia
is strictly aerobic, while
Actinomyces grows in
anaerobic conditions.

Antibiotic susceptibility of
Actinomyces and Nocardia
• Unlike
mycobacteria,
in
nocardiae
and
actinomycetes antibiotic susceptibility can be
tested using diffusion disc test. We have to know,
that they grow slowly and badly.
• For nocardiosis we use co-trimoxazol for therapy,
eventually ampicillin or macrolides.
• In actinomycosis we use penicillin, eventually
doxycyklin and more antibiotics.

Lepromin test in leprosy diagnostics
• There is an animal. Its name is nine banded
armadillo.
• It is necessary for production of lepromin.
• This substance is used in lepromin test, the
equivalent of tuberculin test for TB.

http://www.1costaricalink.com/costa_rica_fauna/nine_band
ed_armadillo.htm

Logo of a TB congress

The End

http://www.niaid.nih.gov